浙江省主要亚热带森林群落类型物种和谱系水平的α和β多样性比较

了解不同森林群落类型的物种和谱系水平的α和β多样性, 有助于指导森林经营和生物多样性保护。本研究比较了浙江省内不同地点主要森林类型(包括常绿阔叶林、常绿落叶阔叶混交林、落叶阔叶林和针阔叶混交林)的物种α多样性和谱系α多样性, 以及物种β多样性和谱系β多样性。研究表明, 该地区主要森林类型的物种和谱系α多样性均存在较大差异, 但控制了空间和地形因子的作用后, 差异几乎全部消失; 森林类型内部及相互间的物种和谱系β多样性均存在显著差异, 同种森林类型内部的物种和谱系β多样性分别小于不同森林类型之间的物种和谱系β多样性, 且在控制了空间和地形因子的作用后, 以上差异仍然显著。本研究表明影响亚热带主要森林群落类型物种和谱系水平的α和β多样性的因素存在差异: α多样性可能主要受到空间和地形因子等的影响, 而β多样性则可能受到森林类型的重要影响。     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 *

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei)TK1501 β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86.63kDa和675.56U/mg。最适作用温度和pH分别为30℃和6.5。 Mg ²⁺和Ca ²⁺对β-葡糖苷酶酶活抑制作用最小,Cu ²⁺几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_max分别为1.44mmol/L和58.32mmol/(L·s),催化系数k_cat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501 β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

黑柄炭角菌(乌灵菌)粉改善睡眠作用以及对小鼠大脑神经递质及其受体的影响

本研究探讨了黑柄炭角菌菌核乌灵菌粉的改善睡眠作用以及对小鼠大脑神经递质及其受体的影响。144例小鼠随机分为低、中、高3个剂量(0.25、0.5和1.0 g/kg bw)的乌灵菌粉实验组和1个空白对照组,进行了30 d的灌胃干预,从小鼠睡眠行为和其大脑组织中神经递质及其受体两个方面进行分析。结果显示,乌灵菌粉无直接催眠作用,对小鼠正常生长和体重增长无显著影响。相比于对照组,乌灵菌粉3个剂量实验组巴比妥钠诱导的入睡潜伏期均显著缩短(P<0.05),戊巴比妥钠阈下剂量下入睡动物数均有增加,戊巴比妥钠诱导的睡眠时间仅有中、高两个剂量组显著增加(P<0.05),低剂量组睡眠时间增加不显著(P>0.05)。低、中、高3个剂量组小鼠大脑组织中γ-氨基丁酸A型受体(GABA_AR)表达量均显著上升(P<0.05),γ-氨基丁酸B型受体(GABA_BR)表达量却显著下降(P<0.05),5-羟色胺(5-HT)和γ-氨基丁酸含量均显著上升(P<0.01)。综上所述,乌灵菌粉具有明显的改善睡眠功效,其作用机制可能是通过提高脑中5-HT和GABA含量,促进小鼠大脑组织中GABA_AR受体表达来实现。     

稀有种和常见种对植物群落物种丰富度格局的相对贡献

物种丰富度格局的形成不仅依赖于群落的构建过程, 同样也依赖于群落中的物种组成(如稀有种和常见种)。本文以黄土高原子午岭林区的辽东栎(Quercus wutaishanica)林为研究对象, 根据频度大小对物种进行排序, 形成稀有-常见种和常见-稀有种两条物种序列, 通过逐一添加(去除)物种, 分析引起的总体物种丰富度及其成分(α多样性和β多样性)的变化, 确定稀有种和常见种对物种丰富度格局的相对贡献。结果表明: (1)常见-稀有种序列与群落总体物种丰富度的相关性呈先剧增后平稳的对数增长曲线, 而稀有-常见种序列与群落总体的相关性与前者刚好相反, 呈先平稳后剧增的指数增长曲线; (2) α多样性在常见-稀有种序列中呈明显的对数变化曲线, 而在稀有-常见种序列中呈指数增长曲线; (3)与α多样性变化相反, β多样性在常见-稀有种序列中随物种的进入先迅速降低后逐渐平稳, 而在稀有-常见种序列中先平稳后急剧降低。可以看出, 常见种不仅主导群落的总体物种丰富度格局, 同时也是α多样性和β多样性格局的重要贡献者。因此, 常见种是群落物种丰富度格局的指示者, 也应该是优先保护的物种。     

迷走神经刺激对难治性癫痫大鼠海马神经炎性反应及α7nAChR表达的影响*

目的: 探讨迷走神经刺激(VNS)对难治性癫痫(IE)模型大鼠海马神经炎性反应及α7nAChR表达的影响。方法: 80只成年雄性SD大鼠,SPF级,随机分为对照组、模型组、VNS组、甲基牛扁亭(MLA)+VNS组,其中对照组与MLA+VNS组分别20只,模型组与VNS组因模型制作失败与动物死亡,分别剩下15只和14只。除对照组之外,其余各组皆通过腹腔注射皮罗卡品建立氯化锂-皮罗卡品IE大鼠模型。对照组仅分离迷走神经,不采取电刺激;模型组不采取任何干预措施;VNS组在模型制作成功后7 d采取VNS,连续4周;MLA+VNS组先侧脑室给药MLA(3.4 μg/μl,5 μl),然后给予VNS,连续4周。观察并记录各组大鼠癫痫发作的次数与持续时间的变化;然后断头处死大鼠,快速分离海马并制备10%组织匀浆,离心并提取上清液,通过分光光度法测定上清液中AChE、ChAT活性;ELISA法检测TNF-ɑ、IL-6和IL-1β表达;Western blot检测海马组织α7nAChR蛋白表达;免疫荧光染色法检测海马组织α7nAChR与小胶质细胞共表达。结果: ①通过VNS治疗4周后,大鼠癫痫发作的频率以及持续的时间都明显低于模型组(P<0.01);MLA阻断后在给予VNS,大鼠癫痫发作的频率以及持续的时间也明显低于模型组,但高于VNS组(P<0.01)。②与对照组比较,模型组大鼠海马组织ChAT表达明显下降,AChE表达明显升高(P<0.01);与模型组比较,VNS组与MLA+VNS组大鼠海马组织ChAT表达明显升高,AChE表达明显降低(P< 0.01);与VNS组比较,MLA+VNS组大鼠海马组织ChAT、AChE表达无明显变化(P>0.05)。③与对照组比较,模型组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显升高(P<0.01);与模型组比较,VNS组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显降低(P<0.01);与VNS组比较,MLA+VNS组大鼠海马组织TNF-ɑ、IL-6和IL-1β表达明显升高(P<0.01)。④与对照组比较,模型组大鼠海马组织以及小胶质细胞上α7nAChR表达明显降低(P<0.01);与模型组比较,VNS组大鼠海马组织以及小胶质细胞上α7nAChR表达明显上调(P<0.01);与VNS组比较,MLA+VNS组海马小胶质细胞上共表达α7nAChR数量明显减少(P<0.01)。结论: VNS对IE大鼠有明显的治疗作用,其机制可能是通过直接激活海马小胶质细胞CAP,抑制海马神经炎性反应来实现的。     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。     

黑河中游春玉米叶片水δD和δ18O的富集过程和影响因素

植物水的稳定同位素分馏过程是水在土壤-植物-大气连续体中循环的重要环节。以往研究由于叶片水¹⁸O同位素比值(δ¹⁸O _l,b)和氘(D)同位素比值(δD_l,b)(合称δ_l,b)实测数量少只能作为模型验证数据, 导致δ_l,b富集机制研究多集中于模型研究, 缺乏基于野外试验条件的δ_l,b富集的控制机制研究。叶片水δD_l,b和δ¹⁸O _l,b的富集程度(ΔD_l,b和Δ¹⁸O _l,b, 合称Δ_l,b)通常表示为δ_l,b与茎秆水D同位素比值(δD_x)和¹⁸O同位素比值(δ¹⁸O_x) (合称δ_x)之差, 即Δ_l,b = δ_l,b - δ_x。该研究以黑河中游沙漠绿洲春玉米(Zea mays)生态系统为研究对象, 重点采集和分析了季节和日尺度δ_l,b和δ_x数据, 配套开展了大气水汽δ¹⁸O和δD (合称δ_v)等辅助变量的原位连续观测, 探讨了季节和日尺度上的δ_l,b富集特征及其影响因素。结果表明: 叶片水δ_l,b和Δ_l,b的季节变化趋势不明显, 而受蒸腾作用影响表现出白天富集夜间贫化的单峰日变化特征。对于D来说, 无论季节尺度上还是日尺度上, 大气水汽δ_v和相对湿度是δD_l,b和ΔD_l,b的主要环境控制因素; 而对于¹⁸O来说, 无论季节尺度上还是日尺度上, 相对湿度是δ¹⁸O _l,b和Δ¹⁸O _l,b的主要环境控制因素。由于D和¹⁸O在热力学平衡分馏上有约8倍差异, 直接分析叶片水ΔD_l,b和Δ¹⁸O_l,b与影响因素的差异性, 有助于理解叶片水δD和δ¹⁸O富集过程以及对模型发展有一定的指导意义。     

毛头鬼伞子实体化学成分及抗糖尿病活性初探

从毛头鬼伞子实体中萃取得到乙醇、乙酸乙酯、石油醚3种有机提取物,采用α-葡萄糖苷酶活性抑制实验对3种有机提取物的抗糖尿病活性进行评价,结果显示,乙酸乙酯提取物对α-葡萄糖苷酶有较强的抑制活性。采用柱层析技术从乙酸乙酯提取物中分离纯化出10种化合物,经核磁等方法鉴定为：（1）顺,顺-9,12-十八(碳)二烯酸;（2）顺式-9-十八烯酸;（3）(22E,24R)-麦角甾烷-5,7,22-三烯-3β醇;（4）3β-5α-6α-22E-麦角甾-7,22-双烯-3,5,6-三醇-6-亚油酸酯;（5）3β-5α-6α-22E-麦角甾-7,22-双烯-3,5,6-三醇-6-油酸酯;（6）邻苯二甲酸二异丁酯;（7）对羟基苯乙醇;（8）4-羟基苯乙基乙酸酯;（9）3-(4-羟基-3-甲氧苯基)败脂酸;（10）N-反式-3,4亚甲二氧基肉桂酰基-3-甲氧基酪胺。对分离化合物的α-葡萄糖苷酶活性抑制实验结果显示,N-反式-3,4亚甲二氧基肉桂酰基-3-甲氧基酪胺对α-葡萄糖苷酶具有较强的抑制活性,其IC₅₀值为4.17mg/mL。     

致幻毒蘑菇卵囊裸盖菇化学成分研究初探

从一种采集于贵州省的致幻毒蘑菇——卵囊裸盖菇Psilocybe ovoideocystidiata中首次分离得到3种化合物,分别是3β-羟基-5α,8α-桥二氧麦角甾-6,22E-二烯(化合物1)、β-D-葡萄糖(化合物2)和腺苷(化合物3)。基于高分辨质谱与核磁共振谱数据以及相关文献比对确定以上3种化合物的结构,并首次推导出化合物2和3质谱裂解规律,其中重排与中性丢失在质谱裂解过程中起主导作用。利用UPLC-MS/MS法对卵囊裸盖菇的干燥子实体和新鲜子实体中的裸盖菇素和脱磷裸盖菇素进行检测,在干燥子实体中检测到裸盖菇素和脱磷裸盖菇素,但在-80 ℃保存6个月的新鲜子实体中未检测到裸盖菇素和脱磷裸盖菇素,推测可能是由于保存方法和提取方法的原因导致化合物发生变化。     

青弋江鱼类分类群和功能群的α和β多样性纵向梯度格局

确定溪流鱼类多样性的时空分布格局可为鱼类多样性保护与管理提供科学基础。尽管溪流鱼类分类群多样性的纵向梯度格局已有大量报道, 但以鱼类生物学特征为基础的功能多样性研究较少。本文基于2009-2010年4个季度对青弋江1-5级溪流共15个样点的调查数据, 利用形态特征数据和食性构建了鱼类复合功能群, 研究了不同级别溪流间鱼类分类群和功能群组成及多样性的异同, 着重探讨了鱼类分类群和功能群的α和β多样性沿溪流纵向梯度的变化规律。采集到的56种鱼类可分为4个营养功能群和5个运动功能群, 共计14个“营养-运动”复合功能群。双因素交互相似性分析结果显示, 鱼类分类群和功能群组成都随河流级别显著变化, 但季节动态不显著; 双因素方差分析后发现, 鱼类分类群和功能群α、β多样性都随河流级别显著变化, 但受季节影响不显著。经回归分析, 分类群和功能群α多样性与河流级别大小呈显著的线性正相关, 但最大分类群α多样性出现于4级河流, 最大功能群α多样性在4级和5级河流间一致; 分类群和功能群β多样性与河流级别大小呈显著的二项式关系, 呈U型分布。分类群β多样性的空间变化主要取决于物种周转, 而功能群β多样性主要由嵌套所驱动。本研究表明, 沿着“上游-下游”的纵向梯度, 河流鱼类的α和β多样性的空间变化规律不同, 分类群和功能群α多样性的空间格局基本一致, 但分类群(主要是物种周转)和功能群β多样性(主要是功能嵌套)的空间变化过程的驱动机制不同。     

The function of Ca channel subtypes in exocytotic secretion: new perspectives from synaptic and non-synaptic release

By mediating the Ca²⁺ influx that triggers exocytotic fusion, Ca²⁺ channels play a central role in a wide range of secretory processes. Ca²⁺ channels consist of a complex of protein subunits, including an ₁ subunit that constitutes the voltage-dependent Ca²⁺-selective membrane pore, and a group of auxiliary subunits, including β, γ, and ₂–δ subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca²⁺ channels are constituted by different combinations of ₁ subunits (of which 10 have been identified) and auxiliary subunits, particularly β (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca²⁺ channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca²⁺ channel structure and function in exocytotic secretion. We discuss Ca²⁺ channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca²⁺ channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca²⁺ channel structure and function is central to our understanding of the fundamental process of exocytotic secretion.     

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein -subunit (G_s), G-protein β₁γ₂ subunit dimer (Gβ₁γ₂), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, G_s, Gβ₁γ₂, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other G_s-coupled receptors together with G_s, Gβ₁γ₂, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, G_s and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.     

Anti-β1 Integrin IgG Inhibits Pulmonary Macrometastasis and the Size of Micrometastases from a Murine Mammary Carcinoma

In the present report, we investigated the possible importance of β₁ integrins in the growth and metastasis of a murine mammary carcinoma, SP1, and a metastatic variant, SP1-3M in vivo. CBA/J female mice bearing SP1 tumor transplants were injected with anti-β₁ integrin IgG or control nonimmune IgG (200 μg per mouse; i.p.) every two days. Animals received anti-CD4 antibody (100 μg per mouse) at time zero to suppress immunity against rabbit IgG. Outgrowth of macroscopic metastases from SP1, but not from SP1-3M primary tumors, was markedly inhibited in animals receiving anti-β₁ integrin IgG but not nonimmune IgG. To assess the stage(s) in the metastatic cascade affected, we examined the number and diameter of micrometastatic nodules in treated and untreated groups. The diameter of micrometastases was significantly reduced in SP1-tumor-bearing mice treated with anti-β₁ integrin IgG compared to control IgG, although the number of nodules per cm² of lung sections examined remained unchanged. No change in the number or size of micrometastases in SP1-3M tumor-bearing mice was observed. No difference in the binding, or complement-mediated and antibody-dependent cell-mediated cytotoxicity of anti-β₁ integrin IgG with SP1 and SP1-3M cells was detected. The results suggest that under these conditions anti-β₁ integrin inhibits metastatic tumor growth in lung tissue, but has minimal effect on intravasation, adhesion to target organs and extravasation.     

Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases.

The horse EE and human β₁β₁ alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp-15 and Trp-314). Tyr-286, which had been proposed to quench the fluorescence of Trp-314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in β₁β₁. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of β₁β₁ is not substantially quenched at alkaline pH. Tyr-286 had also been implicated in the quenching of Trp-314 upon formation of the EE-NAD⁺-trifluoroethanol ternary complex. However, β₁β₁ exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr-286 is not involved in ternary complex quenching.     

Reactivity study of cyclopentadienyl osmium(II) bisphosphine azido complexes with activated alkynes and nitriles: Isolation of osmium triazolato and tetrazolato complexes by 1,3-dipolar addition

The cyclopentadienyl osmium(II) complexes [(η⁵-C₅H₅)Os(PPh₃)₂X] [X = Br (1), CH₃CN (2)] reacts with sodium azide (NaN₃) to yield the corresponding azido complex [(η⁵-C₅H₅)Os(PPh₃)₂N₃] (3). This undergoes [3+2] dipolar cycloaddition reaction with activated alkynes like dimethyl and diethyl acetylenedicarboxylate to yield triazolato complexes [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂(CO₂R)₂}] [R = –CH₂CH₃ (4) and –CH₃ (5)]. The complex 3 also reacts with nitriles such as tetracyanoethylene (TCE), fumaronitrile and p-nitrobenzonitrile to yield complexes of the type [(η⁵-C₅H₅)Os(PPh₃)₂{N₄C₂(CN)C(CN)₂}] (6), [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂HCN}] (7) and [(η⁵-C₅H₅)Os(PPh₃)₂{N₄C(C₆H₄–p-NO₂)}] (8). These complexes were fully characterized on the basis of microanalyses, FT-IR and NMR spectroscopic data. The molecular structure of the representative complex [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂(CO₂CH₂CH₃)₂}] (4) was determined by single crystal X-ray analysis.     

Calcium channel γ subunits provide insights into the evolution of this gene family

The γ subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct γ subunits have been described from human and/or mouse. The first identified member of this group of proteins, γ₁, is a component of the L-type calcium channel expressed in skeletal muscle. A second member, γ₂, identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel γ subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian γ subunits suggests the following relationship ((((γ₂, γ₃), (γ₄, γ₈)), (γ₅, γ₇)), (γ₁, γ₆)) that indicates that they evolved from a common ancestral γ subunit via gene duplication. Our analysis reveals that the novel γ subunit γ₆ most closely resembles γ₁ and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other γ subunits. Rat γ subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of γ₁ mRNA and the long isoform of γ₆ mRNA is most robust in skeletal muscle, while γ₆ is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the γ subunits, we propose that γ₁ and γ₆ may share common physiological functions distinct from the other homologous γ subunits.     

PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching

Recently, it has been shown that PKA-mediated phosphorylation of the β₂-adrenergic receptor (β₂-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G_s and increases its affinity for G_i. Here we demonstrate that, like the β₂-AR, the β₁-AR is also capable of “switching” its coupling from G_s to G_i in a PKA-dependent manner. The β₁-AR is capable of activating adenylate cyclase via G_s, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β₁-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G_i/G_o, and to the PKA inhibitor, H-89. β₁-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G_s-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β₁-AR, like the β₂-AR, can undergo PKA-dependent “G_s/G_i switching”.     

Beta(1)/beta(2)/beta(3)-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet- and cold-induced thermogenesis and of fasting-induced lipolysis, through the β-adrenoceptors (β₁/β₂/β₃). To test this hypothesis, we generated β₁/β₂/β₃-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.     

C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells

GADD34 is a member of a growth arrest and DNA damage (GADD)-inducible gene family. Here, we established a novel Chinese hamster ovary (CHO)-K1-derived cell line, CHO-K1-G34M, which carries a nonsense mutation (termed the Q525X mutation) in the GADD34 gene. The Q525X mutant protein lacks the C-terminal 66 amino acids required for GADD34 to bind to and activate protein phosphatase 1 (PP1). We investigated the effects of GADD34 with or without the Q525X mutation on the phosphorylation status of PP1 target proteins, including the α subunit of eukaryotic initiation factor 2 (eIF2α) and glycogen synthase kinase 3β (GSK3β). CHO-K1-G34M cells had higher levels of eIF2α phosphorylation compared to the control CHO-K1-normal cells both in the presence and absence of endoplasmic reticulum stress. Overexpression of the wild-type GADD34 protein in CHO-K1-normal cells largely reduced eIF2α phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3β phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3β. Cell proliferation rates were higher, while expression levels of the cyclin-dependent kinase inhibitor p21 were lower in CHO-K1-G34M cells compared to the CHO-K1-normal cells. The GADD34 Q525X mutant had a reduced ability to inhibit cell proliferation and enhance p21 expression of the CHO-K1-normal cells compared to the wild-type GADD34 protein. These results suggest that the GADD34 protein C-terminal plays important roles in regulating not only eIF2α dephosphorylation but also cell proliferation in CHO-K1 cells.